Frédéric Geinguenaud

Electrostatic assembly of a DNA superparamagnetic nano-tool for simultaneous intracellular delivery and in situ monitoring

UNLABELLED A superparamagnetic γFe(2)O(3) nanocarrier was developed, characterized by spectroscopic methods and evaluated for the delivery of a decoy oligonucleotide (dODN) in human colon carcinoma SW 480 cells. This nanoparticle-dODN bioconjugate (γFe(2)O(3)@dODN) was designed to target the signal transducer and activator of transcription 3, STAT3, a key regulator of cell survival and proliferation. We exploited a simple precipitation-redispersion mechanism for the direct and one-step complexation of a labeled decoy oligonucleotide with iron oxide nanoparticles (NPs). The cell internalization of the decoy γFe(2)O(3)@dODN nanoparticles is demonstrated and suggests the potential for DNA delivery in biological applications. Despite the increasing use of NPs in biology and medicine, convenient methods to quantify them within cells are still lacking. In this work, taking advantage of the nonlinear magnetic behavior of our superparamagnetic NPs, we have developed a new method to quantify in situ their internalization by cells. FROM THE CLINICAL EDITOR In this study, the authors demonstrate methods to quantify superparamagnetic nanocarriers within cells, taking advantage of the nonlinear magnetic behavior of the studied NPs.

Auto-assembly of E. coli DsrA small noncoding RNA: Molecular characteristics and functional consequences

RNA molecules are important factors involved in different cellular processes and have a multitude of roles in the cell. These roles include serving as a temporary copy of genes used for protein synthesis or functions in translational machinery. Interestingly, RNA is so far the only biological molecule that serves both as a catalyst (like proteins) and as information storage (like DNA). However, in contrast to proteins well known to be able to self-associate in order to maintain the architecture of the cell, such RNA polymers are not prevalent in cells and are usually not favored by the flexibility of this molecule. In this work, we present evidence that such a polymer of a natural RNA, the DsrA RNA, exists in the bacterial cell. DsrA is a small noncoding RNA (87 nucleotides) of Escherichia coli that acts by base-pairing to mRNA in order to control the translation and the turnover of some mRNA, including rpoS mRNA, which encodes the σs RNA polymerase subunit involved in bacterial stress response. A putative model is proposed for the structure of this RNA polymer. Although the function of this polymerization is not known completely, we propose that the formation of such a structure could be involved in the regulation of DsrA ncRNA concentration in vivo or in a quality control mechanism used by the cell to eliminate misfolded RNAs.

Vectorization of Nucleic Acids for Therapeutic Approach: Tutorial Review

Oligonucleotides present a high therapeutic potential for a wide variety of diseases. However, their clinical development is limited by their degradation by nucleases and their poor blood circulation time. Depending on the administration mode and the cellular target, these macromolecules will have to cross the vascular endothelium, to diffuse through the extracellular matrix, to be transported through the cell membrane, and finally to reach the cytoplasm. To overcome these physiological barriers, many strategies have been developed. Here, we review different methods of DNA vectorization, discuss limitations and advantages of the various vectors, and provide new perspectives for future development.

Auto-assembly as a new regulatory mechanism of noncoding RNA

An abstract is not available for this article.

FTIR and UV Spectroscopy Studies of Triplex Formation Between α-Oligonucleotides with Non-Ionic Phoshoramidate Linkages and DNA Targets

Abstract The triplexes formed by pyrimidine α-oligodeoxynucleotides, 15mers αdT15 or 12mers adCT having dimethoxyethyl (PNHdiME), morpholino (PMOR) or propyl (PNHPr) non- ionic phosphoramidate linkages with DNA duplex targets have been investigated by UV and FTIR spectroscopy. Due to the decrease in the electrostatic repulsion between partner strands of identical lengths all modifications result in triplexes more stable than those formed with unmodified phosphodiester β-oligodeoxynucleotides (β-ODNs). Among the α-ODN third strands having C and T bases and non-ionic phosphoramidate linkages (αdCTPN) the most efficient modification is (PNHdiME). The enhanced third strand stability of the αdCTPN obtained as diastereoisomeric mixtures is attenuated by the steric hindrance of the PMOR linkages or by the hydrophobicity of the PNHPr linkages. All αdCTPN strands form triplexes even at neutral pH. In the most favorable case (PNHdiME), we show by FTIR spectroscopy that the triplex formed at pH 7 is held by Hoogsteen T*A.T triplets and in addition by an hydrogen bond between O6 of G and C of the third strand (Tm = 30° C). The detection of protonated cytosines is correlated at pH 6 with a high stabilization of the triplex (Tm = 65° C). While unfavorable steric effects are overcome with a anomers, the limitation of the pH dependence is not completely suppressed. Different triplexes are evidenced for non pH dependent phosphoramidate a- thymidilate strands (adT15PN) interacting with a target duplex of identical length. At low ionic strength and DNA concentration we observe the binding to βdA15 either of αdT15PN as duplex strand and βdT15 as third strand, or of two hydrophobic αdT15PNHPr strands. An increase in the DNA and counterion concentration stabilizes the anionic target duplex and then the αdT15PN binds as Hoogsteen third strand.

Parallel and antiparallel G*G.C base triplets in pur*pur.pyr triple helices formed with (GA) third strands

Abstract Triple helices with G*G.C and A*A.T base triplets with third GA strands either parallel or antiparallel with respect to the homologous duplex strand have been formed in presence of Na+ or Mg2+ counterions. Antiparallel triplexes are more stable and can be obtained even in presence of only monovalent Na+ counterions. A biphasic melting has been observed, reflecting third strand separation around 20°C followed by the duplex -> coil transition around 63°C. Parallel triplexes are far less stable than the antiparallel ones. Their formation requires divalent ions and is observed at low temperature and in high concentration conditions. Different FTIR signatures of G*G.C triplets in parallel and antiparallel triple helices with GA rich third strands have been obtained allowing the identification of such base triplets in triplexes formed by nucleic acids with heterogenous compositions. Only S-type sugars are found in the antiparallel triplex while some N-type sugar conformation is detected in the parallel triplex.

Iron Oxide Nanoparticles Coated with a Phosphorothioate Oligonucleotide and a Cationic Peptide: Exploring Four Different Ways of Surface Functionalization

The superparamagnetic iron oxide nanoparticles (SPIONs) have great potential in therapeutic and diagnostic applications. Due to their superparamagnetic behavior, they are used clinically as a Magnetic Resonance Imaging (MRI) contrast agent. Iron oxide nanoparticles are also recognized todays as smart drug-delivery systems. However, to increase their specificity, it is essential to functionalize them with a molecule that effectively targets a specific area of the body. Among the molecules that can fulfill this role, peptides are excellent candidates. Oligonucleotides are recognized as potential drugs for various diseases but suffer from poor uptake and intracellular degradation. In this work, we explore four different strategies, based on the electrostatic interactions between the different partners, to functionalize the surface of SPIONs with a phosphorothioate oligonucleotide (ODN) and a cationic peptide labeled with a fluorophore. The internalization of the nanoparticles has been evaluated in vitro on RAW 264.7 cells. Among these strategies, the “«one-step assembly»”, i.e., the direct complexation of oligonucleotides and peptides on iron oxide nanoparticles, provides the best way of coating for the internalization of the nanocomplexes.

FTIR and UV Spectroscopy Studies of Triplex Formation Between Pyrimidine Methoxyethylphosphoramidates α- Oligodeoxynucleotides and ds DNA Targets

Abstract The ability of non-ionic methoxyethylphosphoramidate (PNHME) α-oligodeoxynucleotides (ODNs), αdT15 and αdCT dodecamer, to form triplexes with their double-stranded DNA targets was evaluated. Thermal stability of the formed complexes was studied by UV thermal denaturation and the data showed that these PNHME α-ODNs formed much more stable triplexes than phosphodiester (PO) β-ODNs did (ΔTm = + 20° C for αdCT PNHME). In addition, FTIR spectroscopy was used to determine the base pairing and the strand orientations of the triplexes formed by αdT15 PNHME compared to phosphodiester ODNs with β or α anomeric configuration. While βdT15 PO failed to form a triplex with a long βdAn•βdTn duplex, the Tm of the Hoogsteen part of the triplex formed by αdT15 PNHME reached 40° C. Moreover αdT15 PNHME displaced the βdT15 strand of a shorter βdA15•βdT15 duplex. The αdCT PNHME and αdT15 PNHME third strands were found antiparallel in contrast to αdT15 PO which is parallel to the purine strand of their duplex target. The uniform preferential Hoogsteen pairing of the nucleotides αdT and αdC combining both replacements might contribute to the improve stability of the triplexes.

Synthetic melanin bound to subunit vaccine antigens significantly enhances CD8+ T-cell responses

Cytotoxic T-lymphocytes (CTLs) play a key role in immunity against cancer; however, the induction of CTL responses with currently available vaccines remains difficult. Because several reports have suggested that pigmentation and immunity might be functionally linked, we investigated whether melanin can act as an adjuvant in vaccines. Short synthetic peptides (8–35 amino acids long) containing T-cell epitopes were mixed with a solution of L-Dopa, a precursor of melanin. The mixture was then oxidized to generate nanoparticles of melanin-bound peptides. Immunization with melanin-bound peptides efficiently triggered CTL responses in mice, even against self-antigens and at a very low dose of peptides (microgram range). Immunization against a tumor antigen inhibited the growth of established tumors in mice, an effect that was abrogated by the depletion of CD8+ lymphocytes. These results demonstrate the efficacy of melanin as a vaccine adjuvant.